The airway epithelium initiates defenses against inhaled pulmonary pathogens by signaling to recruit phagocytic cells to the site of infection. This signaling is accomplished by toll-like receptors that are activated by conserved bacterial components, stimulate MAPKs and NF-?B signaling cascades to elicit expression of chemokines such as IL-8 that recruit PMNs into the airway. For PMNs to transmigrate across the tight junctions of the epithelial barrier, several membrane spanning proteins of the tight and adherens junctions must be modified. We propose that TLR signaling initiated by TLR2 and possibly TLR5 activates the Ca2+ dependent protease calpain, which targets the epithelial junctional proteins occludin and E-cadherin to facilitate paracellular passage of PMNs into the airway. Similarly, LPS activation of TLR4 induces cleavage of junctional proteins by Ca2+-independent proteases, such as TACE to facilitate PMN transmigration. These same junctional components are also the targets of bacterial virulence factors, the type III secreted toxins of P. aeruginosa, which further modify the epithelial barrier to facilitate bacterial invasion. The combined effects of GTPase inhibition and ADP ribosylation, the activities of P. aeruginosa toxins, alters the localization and functions of junctional proteins critical for the maintenance of the epithelial cytoskeleton and its barrier function. In the experiments proposed we will characterize the signaling process that facilitates PMN migration through the epithelial barrier and how the same targets can be exploited by bacterial pathogens. PUBLIC HEALTH RELEVANCE: Calcium dependent signaling in airway epithelial cells Pneumonia, the accumulation of inflammatory cells in the lung, is a major cause of morbidity and mortality. This project seeks to understand the fundamental process through which polymorphonuclear leukocytes are recruited into the airway in response to bacterial infection.